The overall objective of this project is to determine the necessary protein contributions for binuclear cluster formation and dioxygen binding/activation in binuclear copper proteins. The Dezymer algorithm, which considers on the primary coordination sphere, will be used to predict these binuclear copper metal centers. These centers will be constructed by oligionucleotide directed mutagenesis. Stoichiometry of Cu(II) and Cu(I) binding will be determined. Binuclear cluster formation will be assayed for in all centers. The affinity of these centers for small molecule ligands will be determined. Formation and stability of oxygenated complexes will be determined, in conjunction with the catalytic activity of these complexes. Electronic properties of these centers will be studied to correlate with the formation and stability of the oxygenated complexes. This information will provide the knowledge needed for iterative design rounds for improving oxygen complex stability and catalytic activity of the first design round. By starting with considering the primary coordination sphere and adding non-local interactions successively, I hope to identify the dominant protein interactions that determine the control of oxygen complex stability and reactivity in binuclear copper enzymes.